Cryptococcus neoformans causes a life-threatening human mycoses in severely immunocompromised hosts such as transplant, cancer, and AIDS patients. Despite amphotericin B and newer triazoles for treatment, the management of this infection remains problematic. It is our belief that the discovery of new antifungal drugs coupled with the elicitation of the basic molecular mechanisms by which these drugs act are of prime importance. Over the past few years the molecular biology of C. neoformans has significantly expanded. Effective transformation systems have been developed for C. neoformans and recently in our laboratory gene-knockouts and plasmid rescue procedures have been accomplished using the biolistic DNA delivery system. An animal model of cryptococcal meningitis has been established which closely duplicates the human infection in the central nervous system and has been used both for molecular studies of virulence and treatment evaluations. With this molecular background and the biochemical support of the drug discovery group, we propose to identify and characterize both susceptibility and resistance of C. neoformans to aromatic dicationic compounds at the molecular level. We have found potent fungicidal activity in vitro with some of these compounds and presently we are screening classes of these compounds with the biochemists within the group to identify particular structure-function relationships. Initial molecular studies will focus on topoisomerase I and II of C. neoformans since recent studies indicate that they may be the targets of some dicationic agents. It is probable, however, that some of the new dicationic agents or their derivatives which have potent fungicidal activities in vitro use other molecular targets. Therefore, generation of drug-resistant mutants and restoration of drug susceptibility by gene complementation will be used to determine if there are other molecular targets of these compounds which have high affinity for DNA. It is probable that isolation of genes which restore susceptibility will identify some genes which determine the entrance and/or interaction of the drug with the target molecule. These genes or their deduced proteins will give further structure-function relationships to design selective agents. We expect that by understanding the molecular targets of these drugs through their interaction with C. neoformans DNA we will open up new avenues and chemical designs for our biochemists that may be applicable to other pathogenic fungi. It is of prime importance that antifungal drugs should have as wide spectrum as possible but with low host toxicity. The use of molecular biology for the study of drug targets coupled with the biochemistry of those targeted genes should allow for custom tailoring new drugs in this class.